Fluorescence is the most commonly used nondestructive approach for tracking or analyzing biological molecules in the biosciences. Some luminescent proteins and molecules are known but labeling with luminescent dyes is generally required for optical tracking and analysis of proteins, nucleic acids, lipids, and small molecules. Useful information in the field of bioscience can be obtained by analyzing changes in various chemical and optical properties, including simple changes in luminescent properties, imaging based on changes in luminescent properties, energy transfer, changes in luminescent properties caused by changes in ambient environment, and changes in luminescent properties caused by structural changes via chemical reactions.
Analytical systems for such phenomena include fluorescence microscopes, confocal microscopes, flow cytometers, microarrays, and polymerase chain reaction systems for cell observation, electrophoresis systems for nucleic acid and protein isolation, real-time bioimaging systems, immunoassay systems, DNA sequencing systems, PCR assay systems, diagnostic kits and devices for nucleic acids and proteins, and diagnostic and therapeutic systems such as endoscopes for image-guided surgery. New applications and a number of systems for more accurate and easier analysis are currently under continuous development and improvement.
Suitable fluorescent dyes for use in analytical techniques are required to have high brightness in media, i.e. water-soluble media, where biomolecules exist, stability under various pH conditions, photostability, and excitation and emission wavelength characteristics matched to fluorescence systems. As fluorescent dyes meeting these requirements, Xanthane-based fluoresceins and rhodamines and polymethine-based cyanine derivatives are widely known. Particularly, fluorescent dyes having cyanine chromophores that are widely in use belong to the typical category of dyes.
Presently known carbocyanines with indocarbocyanine, indodicarbocyanine, and indotricarbocyanine skeletons have high molar extinction coefficients but low fluorescence quantum yields. Due to this disadvantage, these carbocyanines were reported to exhibit low brightness after coupled to biomolecules.
This problem needs to be solved to develop novel dyes that can emit stronger fluorescence to obtain effective optical images.
Since 1856, polymethine cyanine dyes have held an unchallenged position in various application fields of dyes. Numerous applications in various areas are being published every year on this subject.
The generic cyanine dyes consist of two nitrogen centers, one of which is positively charged and is linked by a conjugated chain of an odd number of carbon atoms to the other nitrogen. This has been studied as “Push-pull” alkenes and forms the basis of the polymethine dyes, which contain the streptopolymethine unit as the chromophore. Depending upon the charge of the streptomethine unit, these dyes are classified as follows: Cationic streptopolymethine-cyanine and hemicyanine dyes (1), anionic streptopolymethine-oxonol dyes (2), neutral streptopolymethine-merocyanine dyes (3), and zwitterionic squaraine-based cyanine dyes (4):

Generally, the dyes have all-trans geometry in their stable form. Occasionally, these dyes undergo photoisomerization. The formation of these species can be studied by using various techniques such as flash photolysis, transient absorption and picosecond time-resolved spectroscopy. These dyes have been employed extensively as spectral sensitizers in silver halide photography and band-gap semiconductor materials, as recording media in optical discs, in industrial paints for trapping of solar energy, as laser materials, in light-harvesting systems of photosynthesis, as photo-refractive materials, as antitumor agents, and as probes for biological systems.
Generally, the above dyes are deep in color, which is explained by their high molar extinction coefficients, but have the fatal disadvantage of low quantum yield. The reason is that the cyanine dyes in rotational, translational, and vibrational modes lose their excited-state energy by a non-radiative process rather than by a fluorescence process (D. F. O'Brien, T. M. Kelly, and L. F. Costa (1974). Excited state Properties of some Carbocyanine dyes and the energy transfer mechanism of spectral sensitisation. Photogr. Sci. Eng. 18(1), 76-84.). In an attempt to reduce such fluorescence loss, the inventors of the present invention designed cyanine dyes with a rigid polymethine chain to achieve greatly increased fluorescence quantum yield.
Commercially available mitotracker dyes are capable of selective binding to intracellular mitochondria because they are positively charged, are suitable for use as mitotrackers due to their high fluorescence brightness and outstanding photostability, and are sufficiently stained by a simple incubation for labeling of mitochondria.
However, an operation for fixing cells in a reagent, such as formaldehyde, is required in a subsequent experiment. In this operation, however, some commercially available mitotrackers fail to maintain their fluorescence.
The following mitotrackers are commercially available from Thermo Fisher Scientific:

The inventors of the present invention tried to develop dyes whose fluorescence is maintained stable even after fixation and to design dyes emitting fluorescence at various wavelengths so that that a user can choose mitotrackers emitting desired wavelengths.
Materials emitting fluorescence at various wavelengths with narrow bandwidths are considered as important factors because their fluorescence is difficult to analyze when overlapping with the fluorescence wavelengths of other probes. Thus, the inventors of the present invention intended to develop mitotrackers having various wavelengths.
The measurement of intracellular pH or cytosolic pH will greatly facilitate the identification of cellular functions (Methods Mol Biol 637, 311 (2010); Nanotechnology 24, 365 (2013)). On the other hand, many cellular functions, such as ionic homeostasis, reactive oxygen species balance, apoptosis, cell cycle, and cellular mobility, can measured through changes in intracellular pH (Circulation 124, 1806 (2011); Yonsei Med J 6, 473 (1995); J Bacteriol 185, 1190 (2003)).
pH detecting probes are used for the measurement of intracellular pH. Thus, the inventors of the present invention intended to develop dyes whose fluorescence intensity varies in response to pH change and to use the dyes as pH probes capable of measuring the pH of live cells based on this pH-dependent behavior.